Inhibition of 26S and 20S proteasome by indanones

ABSTRACT

This invention is a method for inhibiting cell proliferation using indanones.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention is a method for inhibiting cell proliferation using aclass of indanone compositions never before considered for that purpose.As inhibitors of cell proliferation, the compositions are useful in thetreatment of cancer, cardiovascular disease, e.g. restenosis, host graftrejection, gout, and other proliferative disorders as well as beingpotential therapeutics for autoimmune diseases, such as rheumatoidarthritis, lupus, type I diabetes, multiple sclerosis and similardisorders and diseases.

2. Description of the Art

The multicatalytic proteinase or proteasome is a highly conservedcellular structure that is responsible for the ATP-dependent proteolysisof most cellular proteins. The 20S (700-kDa) proteasome contains atleast five distinct proteolytic activities that have a new type ofmechanism involving a threonine residue at the active site (Coux, O.,Tanaka, K. and Goldberg, A. 1996 Ann. Rev. Biochem. 65:801-47).

The 20S proteasome has been crystallized from the archaebacteriaThermoplasma acidophilum (Lowe, J., Stock, D., Jap. B., Zwickl, P.,Bauminster, W., and Huber, R. 1995 Science 268:533-539). Thearchaebacterial 20S proteasome contains fourteen copies of two distincttypes of subunits, ∝ and β, which form a cylindrical structureconsisting of four stacked rings. The top and bottom rings contain seven∝ subunits each whilst the inner rings contain seven β subunits. A poreextends through the middle of the structure that contains theproteolytic active sites and proteins destined for degradation passthrough this channel. The eukaryotic 20S proteasome is more complex thanthat of the archaebacteria because the number of distinct subunits hasincreased during evolution, however, the subunits can still be classedaccording to the ∝ and β nomenclature of archaebacteria according totheir homology. Thus the quaternary structure of the eukaryotic complexis similar to that of the archaebacteria being comprised of two ∝ andtwo β rings. However, unlike the archaebacterial proteasome thatprimarily exhibits chymotrypsin-like proteolytic activity (Dahlmann, B.,Kopp, F., Kuehn, L., Niedel, B., Pfeifer, G. 1989 FEBS Lett.251:125-131, Seemuller, E., Lupas, A., Zuhl, F., Zwickl, P andBaumeister, W. 1995 FEBS Lett. 359:173; and Lowe, J., Stock, D., Jap,B., Zwickl, P., Bauminster, W. and Huber, R. 1995 Science 268:533-539).The eukaryotic proteasome contains at least five identifiable proteaseactivities. These are termed chymotrypsin-like, trypsin-like andpeptidylglutamyl-peptide hydrolyzing. Two other activities have alsobeen described, one exhibiting a preference for cleavage of peptidebonds on the carboxyl side of branched chain amino acids and the othertoward bonds between small neutral amino acids. (Orlowski, M. 1990Biochemistry 29:10289-10297).

Although the 20S proteasome contains the proteolytic core, it cannotdegrade proteins in vivo unless it is complexed with a 19S cap at eitherend of its structure, which itself contains multiple ATPase activities.This larger structure is known as the 26S proteasome and rapidlydegrades proteins that have been targeted for degradation by theaddition of multiple molecules of the 8.5-kDa polypeptide, ubiquitin.

The first step towards the ubiquitination of a protein proceeds byactivation of a ubiquitin molecule at its carboxyl terminal glycineresidue by addition of ATP that creates a high energy thioesterintermediate. This step is catalyzed by the ubiquitin-activating enzyme,E1. Ubiquitin is then transferred to the active cysteine residue of aubiquitin-conjugating enzyme, E2. E2 enzymes attach ubiquitin to theE-amino groups of lysine residues on the substrate protein that isdestined to be degraded. This process, in some cases also requires aubiquitin ligase, E3. Repeated conjugation of ubiquitin to lysineresidues of formerly bound ubiquitin moieties leads to the formation ofmulti-ubiquitin chains and creates a scaffold of ubiquitin around thesubstrate protein. Multi-ubiquitinated substrate proteins are recognizedby the 26S proteasome and are degraded and the multi-ubiquitin chainsare released from the complex and ubiquitin is recycled.

What causes a protein to become ubiquitinated and thus degraded is stillunder investigation. Clearly this must be a highly regulated series ofevents since the critical timing of specific protein degradation iscrucial for many cell cycle functions. Several signals have beenproposed which largely center upon internal structural sequences withinthe substrate itself. One such proposal is the "N-end rule" in which theamino terminal residue of a protein determines it's half life. Otherproteins such as the cyclins contain a short sequence of highlyconserved amino acids termed the "destruction box" that are apparentlynecessary for degradation. Furthermore "PEST" sequences, which consistof regions rich in proline, aspartate, glutamate, serine and threoninealso seem to act as degradation signals. It is thought that suchinternal sequences act as recognition elements between the proteinsubstrate and its specific ubiquitination machinery.

Two types of inhibitors that inhibit the proteolytic activity of theproteasome, have been described. Certain peptide aldehydes have beenreported to inhibit the chymotrypsin-like activity associated with theproteasome (Vinitsky, A., Michaud, C., Powers, J. and Orlowski, M. 1992Biochemistry 31:9421-9428; Tsubuki, S., Hiroshi, K., Saito, Y.,Miyashita, N., Inomata, M., and Kawashima, S., 1993Biochem.Biophys.Res.Commun. 196:1195-1201; Rock, K, l., Gramm, C.,Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg,A. L. 1994 Cell 78:761-771). These areN-acetyl-L-leucinyl-L-leucinal-L-norleucinal (ALLN) and a closelyrelated compound, N-acetyl-L-leucinyl-L-leucinyl-methional (LLM) with aK_(i) 's of 0.14μ. The most potent inhibitor of this type is astructurally related compound,N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG 115) whichexhibits a K_(i) of 0.021 μM. Although these peptide aldehydes are mosteffective against the chymotrypsin-like proteolytic activity of theproteasomes, careful studies have shown that they are non-specificprotease inhibitors. More recent reports have described series of potentdipeptide inhibitors that have IC₅₀ values in the 10-100 nM range invitro (Iqbal, M., Chatterjee, S., Kauer, J. C., Das, M., Messina, P.,Freed, B., Biazzo, W., and Siman, R. 1995 J.Med.Chem. 38:2276-2277) anda series of similarly potent in vitro inhibitors from ∝-ketocarbonyl andboronic ester derived dipeptides (Iqbal, M., Chatterjee, S., Kauer, J.C., Mallamo, J. P., Messina, P. A., Reiboldt, A., and Siman, R. 1996Bioorg. Med.Chem. Lett 6:287-290).

Another report describes a class of compounds that exhibit specificityin inhibiting proteasome activity (Fenteany, G., Standaert, R. F., Lane,W. S., Choi, S., Corey, E. J., and Schreiber, S. L. 1995 Science268:726-731). Lactacystin is a Streptomyces metabolite that specificallyinhibits the proteolytic activity of the proteasome complex. Thismolecule was originally discovered for its ability to induce neuriteoutgrowth in a neuroblastoma cell line (Omura et al., 1991 J. Antibiot.44:113) later it was shown to inhibit the proliferation of several celltypes (Fenteany et al 1994 Proc.Nat'l. Acad.Sci. USA 91:3358). By usingradiolabelled lactacystin, binding studies by (Fenteany et al., 1995Science 268:726-731) have identified the binding site and the mechanismof action. These studies have shown that lactacystin binds irreversiblyto a threonine residue located at the amino terminus of the β subunit ofthe proteasomes. A series of analogues based upon the structure oflactacystin were also investigated (Fenteany et al., 1995 Science268:726-731). These studies indicated that the β-lactone structure wasessential for its inhibitory activity.

It is now well established that the proteasome is a major extralysosomalproteolytic system which is involved in the degradative pathwaysresulting in numerous and diverse cellular functions such as celldivision, antigen processing and the degradation of short livedregulatory proteins such as transcription factors, oncogene products andcyclins (reviewed in Ciechanover, A. 1994 Cell 79:13-21). The primaryfunction of the proteasome is to catalyze the proteolysis of proteinsinto small peptides. However, it has also been demonstrated that theubiquitin-proteasome pathway can catalyze the regulated proteolyticprocessing of a large inactive precursor into an active protein. Thebest documented case of this involves the activation of thetranscription factor NF-κB (Palombella, V. J., Rando, O. J., Goldberg,A. L., and Maniatis, T. 1994 Cell 78:773-785). The active form of NF-κBis a heterodimer consisting of a p65 and a p50 subunit. The latter ispresent in the cytosol of the cell in an inactive precursor form, namelyp105, the 105-kDa polypeptide precursor of p50. The proteolyticprocessing of p105 to generate p50 occurs via the ubiquitin-proteasomepathway. Additionally, processed p50 and p65 is maintained in thecytosol as an inactive complex with the inhibitory protein IκB.Inflammatory signals activate NF-κB by initiating the signalling pathwayfor the complete degradation of IκB, and also stimulate the processingof p105 into p50. Thus two proteolytic events, both governed by theubiquitin-proteasome pathway, are required for signal induced activationof NF-κB. What causes the termination of proteolysis of p105 followingveneration of p50 is not known but it has been proposed that theconformation of p50 may render it resistant to further processing andcause it to dissociate from the 26S complex.

The fact that the proteasome plays a critical event in the activation ofNF-κB could be exploited clinically by the use of inhibitors directedtowards proteasome proteolysis. In certain diseases the normal functionof active NF-κB can be detrimental to human health as observed ininflammatory responses following bacterial, fungal or viral infection.Thus inhibitors of NF-κB activation, due to their ability to preventsecretion of cytokines, may have potential utility in the treatment ofARDS (acute respiratory distress syndrome) and AIDS. Since activation ofNF-κB is also essential for angiogenesis, proteasome inhibitors may haveutility in the treatment of those diseases associated with abnormalneovascularization.

p53 was first described as an oncoprotein but has since been shown to beinvolved in many cellular processes (reviewed by Ko, L. J. and Proves,C. 1996 Genes Dev. 10, 1054-1072). p53 has been shown to induceapoptosis in several haematopoietic cell lines (Oren, M., 1994 Semin.Cancer Biol. 5, 221-227) through the action of many different stimuliincluding DNA damage, viral infection and the removal of growth factors.However, it is important to note that apoptosis can be induced in ap53-independent manner for example by the action of glucocorticoids.Induction of p53 leads to cell growth arrest in the G1 phase of the cellcycle as well as cell death by apoptosis. Both of these functions allowp53 to control DNA damage thereby reducing the propagation of DNAmutations when cells divide. p53 arrests cells at G1 by inducing thecyclin-dependent kinase inhibitor, p21, which in turn causes anaccumulation of the hypophosphorylated form of the retinoblastoma geneproduct. It is thought that p53 acts as a check point in the cellfollowing DNA damage, it first causes an arrest in cell division andapoptosis. p53 degradation is known to be via the ubiquitin-proteasomepathway and disrupting p53 degradation is a possible mode of inducingapoptosis. Another potential utility of proteasome inhibitors may be inthe treatment of diseases that result from abnormal cell proliferation.

It is well documented that the ubiquitin-proteasome pathway is criticalfor the regulated destruction of cyclins that govern the exit frommitosis and allow cells to progress into the next phase of the cellcycle. Thus inhibiting degradation of cyclins by using proteasomeinhibitors causes growth arrest. Therefore another potential utility ofproteasome inhibitors is their use in the treatment of diseases thatresult from an accelerated cell division. These include cancer,cardiovascular diseases such as myocarditis, restenosis followingangioplasty, renal diseases such as lupus, polycystic kidney disease,fungal infections, dermatological diseases such as psoriasis, abnormalwound healing, keloids, immunological diseases such as autoimmunity,asthma, and allergy, acute and delayed hypersensitivity, graft versushost disease, transplant rejection and neuroimmunological diseases suchas multiple sclerosis and acute disseminated encephalomyelitis.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a method for inhibitingcell proliferation in mammals that uses a therapeutically effectiveamount of a composition heretofore unknown for its cell proliferativeinhibition properties.

It is an object of this, invention to provide a method for the effectivetreatment of diseases that result from accelerated cell division.

It is another object of this invention to provide a method for thetreatment of proliferative diseases that operates by inhibiting thedegradation of proteasome inhibitors.

It is another object of this invention to use a therapeuticallyeffective amount of the composition to inhibit cell proliferativedisorders in humans.

In one embodiment, this invention is a method for inhibiting cellproliferation in mammals comprising administering to the mammal atherapeutically effective amount of a compound having the formula:##STR1##

In the compound, R₁ -R₄ are each individually selected from the groupincluding hydrogen, halogen, hydroxyl, thiol, lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocycle, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

R₅ -R₉ are each individually selected from the group of compoundsincluding hydrogen, halogen, hydroxyl, thiol, oxo, lower alkyl,substituted lower alkyl, alkenyl, alkynyl, alkylalkenyl, alkyl alkynyl,alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocycle,heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano;

X is selected from the group of compounds including hydrogen,-D₁,-D₂,-E, -D₁ -E, -D₂ -E, -D₁,-D₂, or a compound having the formula: ##STR2##wherein D₁, and D₂ are each individually selected from the group ofcompounds including a compound having the formula: ##STR3## or hydrogen,halogen, hydroxyl, thiol, lower alkyl, substituted lower alkyl, alkynyl,alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocyclic, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, or alkylcycloheteroalkyl;

wherein E is selected from the group of compounds including: ##STR4## orhydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl,aryloxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocycle,heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, oralkylcycloheteroalkyl.

When D₁, D₂ and/or E are selected from compounds including substituentsR₁₀ -R₁₄, J₁ and J₂, then R₁₀ -R₁₄ are each individually selected fromthe group of compounds including hydrogen, halogen, hydroxyl, thiol,oxo, lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, or cyano andJ₁ and J₂ are each individually selected from the group of compoundsincluding N--R₁₅, CR₁₆ R₁₇, O, S--(O)₀₋₂, P--(O)₀₋₃, wherein R₁₅ -R₁₇may each be individually selected from the group of compounds includinghydrogen, halogen, hydroxyl, oxo, thiol, lower alkyl, substituted loweralkyl, alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl,aryloxy, amino, amido, carboxyl, aryl, substituted aryl, heterocyclic,heteroaryl, substituted heterocycle, heteroalkyl, cycloalkyl,substituted cycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, orcyano.

The compositions are useful, when administered in therapeutic amounts,for treating mammals, and preferably for treating humans suffering fromcell proliferative disorders, infectious diseases, and immunologicaldiseases.

DESCRIPTION OF THE FIGURES

In the Figures, FIG. 1 is a Western Blot immunoreactivity assay using ananti-IκBα antibody of RAW cell extract which had been treated withcompounds 173 and 187 which are described in Tables 1 and 2;

FIG. 2 is a Western Blot immunoreactivity assay towards an anti-P50antibody of RAW cell extracts which had been treated with compound 187which is described in Tables 1 and 2 prior to exposure to its LPS; and

FIG. 3 is a gel mobility shift assay using nuclear extract prepared fromRAW cells that had been pretreated compound 187 which is described inTables 1 and 2 prior to exposure to LPS.

DESCRIPTION OF THE CURRENT EMBODIMENT

This invention is a method for inhibiting cell proliferation disorders,infectious diseases, and immunological diseases in mammals andespecially in humans using compositions having the following generalformula: ##STR5##

In the composition, R₁,-R₄ are each individually selected from the groupof compounds including hydrogen, halogen, hydroxyl, thiol, lower alkyl,substituted lower alkyl, alkenyl, alkynyl, alkylalkenyl, alkyl alkynyl,alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocycle,heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

In the composition, R₅ -R₉ are each individually selected from the groupof compounds including hydrogen, halogen, hydroxyl, thiol, oxo, loweralkyl, substituted lower alkyl, alkenyl, alkynyl, alkylalkenyl, alkylalkynyl, alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocycle,heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, nitro, or cyano.

X is selected from the group of compounds including hydrogen, -D₁, -D₂,-E, -D₁ -D₂, -D₁ -E₁, -D₂ -E or a compound having the formula: ##STR6##wherein D₁ and D₂ are each each individually selected from the group ofcompounds including ##STR7## hydrogen, halogen, hydroxyl, thiol, loweralkyl, substituted lower alkyl, alkynyl, alkylalkenyl, alkyl alkynyl,alkoxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocyclic,heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, oralkylcycloheteroalkyl, and

wherein E is selected from the group of compounds including: ##STR8##hydrogen, halogen, hydroxyl, thiol, lower alkyl, substituted loweralkyl, alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl,aryloxy, alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl,substituted aryl, heterocycle, heteroaryl, substituted heterocycle,heteroalkyl, cycloalkyl, substituted cycloalkyl, alkylcycloalkyl, oralkylcycloheteroalkyl.

In the compounds identified above, R₅ -R₉ are each individually selectedfrom the group of compounds including hydrogen, halogen, hydroxyl,thiol, oxo, lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, or cyano.

When D₁, D₂ and/or E are selected from compounds including substituentsR₁₀ -R₁₄ J₁ and J₂, then: R₁₀ -R₁₄ are each individually selected fromthe group of compounds including hydrogen, halogen, hydroxyl, thiol,oxo, lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkylalkenyl, alkyl alkynyl, alkoxy, alkylthio, acyl, aryloxy, amino,amido, carboxyl, aryl, substituted aryl, heterocycle, heteroaryl,substituted heterocycle, heteroalkyl, cycloalkyl, substitutedcycloalkyl, alkylcycloalkyl, alkylcycloheteroalkyl, nitro, or cyano; andJ₁ and J₂ are each individually selected from the group includingN--R₁₅, CR₁₆ R₁₇, O, S--(O)₀₋₂, P--(O)₀₋₂, wherein R₁₅ -R₁₇ are eachindividually substituted with a component selected from the groupincluding hydrogen, halogen, hydroxyl, oxo, thiol, lower alkyl,substituted lower alkyl, alkynyl, alkylalkenyl, alkyl alkynyl, alkoxy,alkylthio, acyl, aryloxy, amino, amido, carboxyl, aryl, substitutedaryl, heterocyclic, heteroaryl, substituted heterocycle, heteroalkyl,cycloalkyl, substituted cycloalkyl, alkylcycloalkyl,alkylcycloheteroalkyl, or cyano.

The following term are used to describe various constituents of thechemical composition useful in the method of this invention. The termsare defined as follows:

The term "halogen" refers to fluorine, bromine, chlorine, and iodineatoms.

The term "hydroxyl" refers to the group --OH.

The term "oxo" refers to the group =0.

The term "thiol" or "mercapto" refers to the group --SH, and --S(O)₀₋₂.

The term "lower alkyl" refers to a cyclic, branched or straight chain,alkyl group of one to ten carbon atoms. This term is further exemplifiedby such groups as methyl, ethyl, n-propyl, I-propyl, n-butyl, t-butyl,l-butyl (or 2-methylpropyl), cyclopropylmethyl, I-amyl, n-amyl, hexyland the like.

The term "substituted lower alkyl" refers to lower alkyl as justdescribed including one or more groups such as hydroxyl, thiol,alkylthiol, halogen, alkoxy, amino, amido, carboxyl, cycloalkyl,substituted cycloalkyl, heterocycle, cycloheteroalkyl, substitutedcycloheteroalkyl, acyl, carboxyl, aryl, substituted aryl, aryloxy,hetaryl, substituted hetaryl, aralkyl, heteroaralkyl, alkyl alkenyl,alkyl alkynyl, alkyl cycloalkyl, alkyl cycloheteroalkyl, cyano. Thesegroups may be attached to any carbon atom of the lower alkyl moiety.

The term "alkenyl" refers to a group --CR'═CR"R"' where R', R", R"' areeach selected from hydrogen, halogen, lower alkyl, substituted loweralkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroarylor the like as defined.

The term "alkynyl" refers to a group --C.tbd.C--R'; where R' is selectedfrom hydrogen, halogen, lower alkyl, substituted lower alkyl, acyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl or the likeas defined.

The term "alkyl alkenyl" refers to a group --R--CR'═CR"'R"", where R islower alkyl, or substituted lower alkyl, R', R"', R"" are eachindependently selected from hydrogen, halogen, lower alkyl, substitutedlower alkyl, acyl, aryl, substituted aryl, hetaryl, or substitutedhetaryl as defined below.

The term "alkyl alkynyl" refers to a group --RC.tbd.--CR' where R islower alkyl or substituted lower alkyl, R' is hydrogen, lower alkyl,substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, orsubstituted hetaryl as defined below.

The term "alkoxy" refers to the group --OR, where R is lower alkyl,substituted lower alkyl, acyl, aryl, substituted aryl, aralkyl,substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, or substitutedcycloheteroalkyl as defined below.

The term "alkylthio" denotes the group --SR, --S(O)_(n=1-2) --R, where Ris lower alkyl, substituted lower alkyl, aryl, substituted aryl aralkylor substituted aralkyl as defined below.

The term "acyl" refers to groups --C(O)R, where R is hydrogen, loweralkyl substituted lower alkyl, aryl, substituted aryl and the like asdefined below.

The term "aryloxy" refers to groups --OAr, where Ar is an aryl,substituted aryl, heteroaryl, or substituted heteroaryl group as definedbelow.

The term "amino" refers to the group NRR', where R and R' mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl, hetaryl, cycloalkyl, or substituted hetaryl as definedbelow or acyl.

The term "amido" refers to the group --C(O)NRR', where R and R' mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl, hetaryl, substituted hetaryl as defined below.

The term "carboxyl" refers to the group --C(O)OR, where R mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl, hetaryl, substituted hetaryl and the like as defined.

The terms "aryl" or "Ar" refer to an aromatic carbocyclic group havingat least one aromatic ring (e.g., phenyl or biphenyl) or multiplecondensed rings in which at least one ring is aromatic, (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl).

The term "substituted aryl" refers to aryl optionally substituted withone or more functional groups, e.g., halogen, lower alkyl, lower alkoxy,lower alkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl,aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano,alkylthio, thiol, sulfamido and the like.

The term "heterocycle" refers to a saturated, unsaturated, or aromaticcarbocyclic group having a single ring (e.g., morpholino, pyridyl orfuryl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl,quinolinyl, indolizinyl or benzo b!thienyl) and having at least onehetero atom, such as N, O or S, within the ring, which can optionally beunsubstituted or substituted with, e.g., halogen, lower alkyl, loweralkoxy, lower alkylthio, trifluoromethyl, amino, amido, carboxyl,hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl,nitro, cyano, alkylthio, thiol, sulfamido and the like.

The terms "heteroaryl" or "hetar" refer to a heterocycle in which atleast one heterocyclic ring is aromatic.

The term "substituted heteroaryl" refers to a heterocycle optionallymono or poly substituted with one or more functional groups, e.g.,halogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl,amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl,substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and thelike.

The term "aralkyl" refers to the group --R--Ar where Ar is an aryl groupand R is lower alkyl or substituted lower alkyl group. Aryl groups canoptionally be unsubstituted or substituted with, e.g., halogen, loweralkyl, alkoxy, alkyl thio, trifluoromethyl, amino, amido, carboxyl,hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl,nitro, cyano, alkylthio, thiol, sulfamido and the like.

The term "heteroalkyl" refers to the group --R--Het where Het is aheterocycle group and R is a lower alkyl group. Heteroalkyl groups canoptionally be unsubstituted or substituted with e.g., halogen, loweralkyl, lower alkoxy, lower alkylthio, trifluoromethyl, amino, amido,carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substitutedhetaryl, nitro, cyano, alkylthio, thiol, sulfamido and the like.

The term "heteroarylalkyl" refers to the group --R--HetAr where HetAr isan heteroaryl group and R lower alkyl or substituted loweralkyl.Heteroarylalkyl groups can optionally be unsubstituted or substitutedwith, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy,alkylthio, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl,nitro, cyano, alkylthio, thiol, sulfamido and the like.

The term "cycloalkyl" refers to a divalent cyclic or polycyclic alkylgroup containing 3 to 15 carbon For polycyclic groups, these may bemultiple condensed rings in which one of the distal rings may bearomatic (e.g. indanyl, tetrahydronaphthalene, etc . . . ).

The term "substituted cycloalkyl" refers to a cycloalkyl groupcomprising one or more substituents with, e.g., halogen, lower alkyl,substituted lower alkyl, alkoxy, alkylthio, aryl, aryloxy, heterocycle,hetaryl, substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamidoand the like.

The term "cycloheteroalkyl" refers to a cycloalkyl group wherein one ormore of the ring carbon atoms is replaced with a heteroatom (e.g., N, O,S or P).

The term "substituted cycloheteroalkyl" refers to a cycloheteroalkylgroup as herein defined which contains one or more substituents, such ashalogen, lower alkyl, lower alkoxy, lower alkylthio, trifluoromethyl,amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl,substituted hetaryl, nitro, cyano, alkylthio, thiol, sulfamido and thelike.

The term "alkyl cycloalkyl" refers to the group -R-cycloalkyl wherecycloalkyl is a cycloalkyl group and R is a lower alkyl or substitutedlower alkyl. Cycloalkyl groups can optionally be unsubstituted orsubstituted with e.g. halogen, lower alkyl, lower alkoxy, loweralkylthio, trifluoromethyl, amino, amido, carboxyl, hydroxyl, aryl,aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano,alkylthio, thiol, sulfamido and the like.

The term "amino acid" refers to the D- or L-isomer of naturallyoccurring and synthetic alpha amino acids preferably the amino acids arenaturally occurring alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histaidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine or valine.

Typically D₁, D₂ and E, if present in the composition, may be aminoacids. Usually lipophilic amino acids are preferred. In general, theamino acid abbreviations follow the IUPAC-IUB Joint Commission onBiochemical Nomenclature as described in Eur. J. Biochem, 158 9(1984).

It is preferred that R3 is methoxy, -D₁ is leucine, and -D₂ is leucine,and E is NR'R". It is most preferred that -D₁, is 1-leucine, and -D₂ isd-leucine and that E is selected from the group of compounds consistingof benzylamine, 1-indanylamine, N,N'-dibenzylamine,2,6-difluorobenzylamine, 4-methoxybenzylamine, piperonyl amine, NH₂ andglycineamide.

In one preferred composition, R₃ is methoxy D₁, is leucine D₂ isleucine, and E is benzylamine. In another preferred composition, R₃ ismethoxy, D₁ is leucine, D₂ is leucine, and E is 1-indanylamine. In stillanother preferred composition, R₃ is methoxy, D₁ is leucine, D₂ isleucine, and E is N,N-dibenzylamine. In another preferred composition,R₃ is methoxy, D₁ is leucine, D₂ is leucine, and E is2,6-difluorobenzylamine.

In these preferred compositions, it is further preferred that D₁, isL-Leucine and is D₂ is D-Leucine. Known compounds that may be useful inthe therapeutic method of this invention are disclosed in Table 1immediately below.

                                      TABLE 1                                     __________________________________________________________________________    R1 R2 R3 R4 R5   R6    R7                                                                              R8     R9    D1                                      __________________________________________________________________________       Cl       Me                        OH                                            OMe                                                                              OMe             ═N--OH   OMe                                                 Ph                        Me                                         Cl       Me           ═N--OH   OEt                                        OMe                                OH                                                               Me           OMe                                     F     F                               NH2                                                                           NHEt                                                     Ph      ═CHPh    OH                                            F                               OH                                                                            OH                                            OMe   Me                        OH                                         Me       Me                        OH                                                  Ph           ═N--OH   OMe                                           OMe                ═CH-(4-  OH                                                               dimethylamin                                                                  ophenyl)                                                OMe                   ═N--OH   OMe                                                 Ph           COOEt        Me                                                  Me           ═N--OH   OEt                                           Me                              OMe                                                      Me                   OH                                         Cl       Me           NHCOCOOEt    OEt                                           OMe                                                                              OMe                          OH                                                  Me                        Me                                      F     F                               OH                                                                            NHMe                                                Ph                        OH                                            OMe   Me           ═N--OH   OMe                                           OMe                             OH                                                  Me           NHAc         OMe                                                      Ph      Ph           OH                                                  Ph                        NHCyclopropyl                              OMe                                                                              OMe                             OH                                                  Ph                        OH                                                  COOMe                     OMe                                        OMe                                                                              OMe                ═N--OH   OMe                                                 Ph                        OMe                                        Cl       Me                        OEt                                           OMe                                                                              OME                          OMe                                                              Me           OH                                                  Me           NHCOCOOEt    OEt                                                                           NH-n-propyl                             OMe      NO2                          OH                                            F                               Cl                                            OMe                             OMe                                           OMe   Me           ═N--OH   OMe                                           OMe   Me                        OMe                                        Me       Me                        OMe                                           F                  Me           OH                                                       4-Methoxy            OH                                                       phenyl                                                                                ═N--OH   OMe                                                 Ph   CN      COOEt        OEt                                                 Me                        OH                                         OMe                                OMe                                           OMe                             4-  4-(1-                                                                     methylethyl)-1-                                                               piperazinyl!car                                                               bonyl!phenoxy                              Cl       Me           NH2          OEt                                           Me                              OH                                                               Me           OH                                      F     F                               Cl                                                                            OMe                                                 Ph                        OH                                            OMe                ═N--OH   OMe                                                      2-naphthyl           OH                                            OMe   Me                        OMe                                                      Ph      Ph           OMe                                                 Ph                        NH-n-propyl                                   Me                 ═N--OH   OMe                                                      Ph                   OH                                            OMe                ═N--OH   OMe                                                 Ph   SH      COOEt        OH                                         Cl       Me                        OH                                         OMe                                                                              OMe                             OMe                                                      Me                   OH                                                  Me           NH2          OEt                                                                           NH-n-Butyl                                 NO2                                                                              OMe                             OH                                            F                               NH2                                                              Me           OH                                                  Ph                        OH                                            OMe                             OMe                                        Me       Me           3-propanoic                                                                          3-propanoic                                                                         OH                                                               acid   acid                                                      Me                        OMe                                        OMe           Ph                   OH                                            OMe   Ph                        NHMe                                                Me           ═N--OH   OMe                                                 Ph   COOEt   COOEt        OEt                                           OMe   COOMe                     OMe                                     __________________________________________________________________________

It is within the knowledge of one of skill in the art that stereoisomersof the compositions described herein as well as isomer and stereoisomersof components that comprise the compositions identified herein all fallwithin the scope of compositions that are useful in the therapeuticmethod of this invention.

If the compound useful in the method of this invention contains a basicgroup, an acid addition salt may be prepared. Acid addition salts of thecompounds are prepared in a standard manner in a suitable solvent fromthe parent compound and an excess of acid, such as hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic, ormethanesulfonic. If the final compound contains an acidic group,cationic salts may be prepared. Typically the parent compound is treatedwith an excess of an alkaline reagent, such as hydroxide, carbonate oralkoxide, containing the appropriate cation. Cations such as NA⁺, K⁺,Ca⁺² and NH⁺⁴ are examples of cations present in pharmaceuticallyacceptable salts. Certain of the compounds form inner salts orzwitterions which may also be acceptable.

The compounds described above are useful for treating cell proliferationdisorders, infectious diseases, and immunological diseases in mammals,and specifically, in human patients who require such treatment. Cellproliferative disorders that may be treated using the compositiondescribed above include cancer, cardiovascular disease such asmyocarditis and restenosis following angioplasty, renal diseases such aslupus and polycystic kidney disease, host graft rejection, gout, andother proliferative disorders. Autoimmune diseases that may be treatedwith the compositions described above include rheumatoid arthritis,lupus, type I diabetes, multiple sclerosis and similar disorders anddiseases. Infectious diseases that may be treated using the compositionsdescribed above include IBD, Crohn's disease, AIDS, ARDS, and similardisorders. The compositions disclosed above can also be used to treatfungal infections, dermatological diseases such as psoriasis, abnormalwound healing, keloids, immunological diseases such as autoimmunity,asthma, allergy, acute and delayed hypersensitivity, graft versus hostdisease, transplant rejection, and neuroimmunological disease such asmultiple sclerosis and acute disseminated encephalomyelitis.

The method of treatment of these diseases and disorders comprises theadministration parenterally, or orally of an effective quantity of thechosen compound or combinations thereof, preferably dispersed in apharmaceutical carrier. Dosage units of the active ingredient aregenerally selected from the range of 0.01 to 100 mg/kg, but will bereadily determined by one skilled in the art depending upon the route ofadministration, age and condition of the patient. These dosage units maybe administered one to ten times daily for acute or chronic disease. Nounacceptable toxicological effects are expected when compounds of theinvention are administered in accordance with the present invention.

Pharmaceutical compositions of the compounds of this invention, orderivatives thereof, may be formulated as solutions or lyophilizedpowders for parenteral administration. Powders may be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation is generally a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, standard 5% dextrose in water or bufferedsodium or ammonium acetate solution. Such formulation is especiallysuitable for parenteral administration, but may also be used for oraladministration. It may be desirable to add excipients such aspolyvinylpyrrolidinone, gelatin, hydroxycellulose, acacia, polyethyleneglycol, mannitol, sodium chloride or sodium citrate. Alternatively,these compounds may be encapsulated, tableted or prepared in an emulsionor syrup for oral administration. Pharmaceutically acceptable solid orliquid carriers may be added to enhance or stabilize the composition, orto facilitate preparation of the composition. Liquid carriers includesyrup, peanut oil, olive oil, glycerin, saline, alcohols and water.Solid carriers include starch, lactose, calcium sulfate, dehydrate,terra alba, magnesium stearate or stearic acid, talc, pectin, acacia,agar or gelatin. The carrier may also include a sustained releasematerial such as glyceryl monostearate or glyceryl distearate, alone orwith a wax. The amount of solid carrier varies but, preferably, will bebetween about 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulation, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is use, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule.

EXAMPLE 1

The compounds useful in the therapeutic method of this invention areprepared by conventional methods of organic chemistry. Coupling reagentsare well known in the art, such as DCC and other carbodiirnides, EDC,BOP and PPA, and they may optionally be used with other reagents, suchas HOBT, NMM and DMAP, which may facilitate the reaction. Preparation ofcompounds of the formula (1) wherein D₁, D₂ and E are amino acids arewell known in the art using either conventional solution phase or solidphase techniques as described in Bodanszky, "The Practice of PeptideSynthesis," Springer-Verlag, First Edition, 1984. Suitable protectivegroups for the amino group are those disclosed by Greene et al.,"Protective Group in Organic Synthesis," Second Edition, John Wiley andSons, N.Y., 1991. The benzyloxycarbonyl, t-butoxycarbonyl andfluorenylmethoxycarbonyl groups are especially useful amino protectinggroups.

Solid phase peptide synthesis was accomplished as follows. Rink amideresin is placed in a syringed fitted with a fritted filter. The resin isdeprotected using 20% piperidine in DMF. After 20 minutes, the resin waswashed five times with DMF, five times with methanol, then five timeswith DMF. A solution of amino acid (E), carbodiimide and HOBT in DMF wasdrawn up into the syringe and the reaction mixture was allowed to mixfor 4 to 20 hours. The reaction solution was ejected and the mixturewashed five times with DMF, five times with methanol, then five timeswith DMF. This cycle was repeated until the desired sequence wasattached. The final coupling used, 5-methoxy-1-indanone-3-acetic acid,carbodiimide and HOBT. After final washings of the resin, the peptidefragment was cleaved from the resin using 95% TFA/5% water.Concentration of the cleavage mixture affords a white solid.

EXAMPLE 2

Compounds of this invention prepared according to the method of Example1 were tested as follows. The 20S catalytic subunit of the proteasome(also known as the multicatalytic proteinase complex) was purified tohomogeneity from bovine brain according to published methods (Wilk S.and Orlowski, M, 40 842 J. Neurochem (1983)). The chymotryptic activityof the complex is measured by the increase in fluorescence followingcleavage of the substrate peptidesuccinyl-leucine-leucine-valine-tyrosine-7-amino-4-methyl coumarin. Thestandard in vitro assay consists of 2 μg 20S proteasome, 0.1-100 μg/mlproteasome inhibitor in 200 μl 50 mM HEPES, containing 0.1% sodiumdodecyl sulphate, pH7.5. The proteolytic reaction is initiated by theaddition of 50 μM fluorogenic peptide substrate and allowed to progressfor 15 minutes at 37° C. The reaction is terminated by the addition of100 μl of 100 mM acetate buffer, pH4.0. The rate of proteolysis isdirectly proportional to the amount of liberated aminomethylcoumarinwhich is measured by fluorescent spectroscopy (EX 370 nm, EM 430 nm).The structures of the compounds tested as well as the test results arereported in Table 2, below.

                                      TABLE 2                                     __________________________________________________________________________                                                   IC50                           Compound                                                                            R3 Dl        D2            E             ug/ml                          __________________________________________________________________________    1     MeO                                                                              d-leu--NH2                            5                              2     MeO                                                                              d-leu     d-leu--NH2                  1                              3     MeO                                                                              leu--NH2                              >10                            4     MeO                                                                              d-leu     his--NH2                    >10                            5     MeO                                                                              leu       leu--NH2                    >10                            6     MeO                                                                              d-leu     leu--NH2                    >10                            7     MeO                                                                              leu       d-leu--NH2                  >10                            8     MeO                                                                              Nle--NH2                              >10                            9     MeO                                                                              d-Nle--NH2                            >10                            10    MeO                                                                              Nva--NH2                              >10                            11    MeO                                                                              d-leu     Aib--NH2                    >10                            12    MeO                                                                              d-phe--NH2                            >10                            13    MeO                                                                              CHA       Leu--NH2                    >10                            14    MeO                                                                              phe       leu--NH2                    >10                            15    MeO                                                                              Nle       d-Leu--NH2                  >10                            16    MeO                                                                              Nle       leu--NH2                    >10                            17    MeO                                                                              leu       leu           gly--NH2      >10                            18    MeO                                                                              d-leu     leu           gly--NH2      5                              19    MeO                                                                              d-leu     d-leu         gly--NH2      >10                            20                                                                            21    MeO                                                                              leu       d-leu         gly--NH2      0.1                            22    MeO                                                                              d-Nle     d-leu--NH2                  >10                            23    MeO                                                                              d-Nle     leu--NH2                    >10                            24    MeO                                                                              phe--NH2                              >10                            25    MeO                                                                              Tic--NH2                              >10                            26    MeO                                                                              Tic       d-leu--NH2                  >10                            27    MeO                                                                              d-phe     d-leu--NH2                  10                             28    MeO                                                                              leu       Aib--NH2                    >10                            29    MeO                                                                              CHA--NH2                              >10                            30    MeO                                                                              d-val     d-leu--NH2                  >10                            31    MeO                                                                              d-pro     d-leu--NH2                  10                             32    MeO                                                                              d-cha     d-leu--NH2                  >10                            33    MeO                                                                              d-leu     d-val--NH2                  >10                            34    MeO                                                                              d-val     d-val--NH2                  >10                            35    MeO                                                                              d-cha     d-cha--NH2                  >10                            36    MeO                                                                              d-phg--NH2                            >10                            37    MeO                                                                              d-phg     d-leu--NH2                  >10                            38    MeO                                                                              benzyl                                >10                            39    MeO                                                                              dibenzylamine                         >10                            40    MeO                                                                              gly       gly           gly--NH2      >10                            41    MeO                                                                              leu       gly           gly--NH2      >10                            42    MeO                                                                              gly       d-leu         gly--NH2      >10                            43    MeO                                                                              nle       d-leu         gly--NH2      >10                            44    MeO                                                                              nva       d-leu         gly--NH2      >10                            45    MeO                                                                              phe       d-leu         gly--NH2      >10                            46    MeO                                                                              cha       d-leu         gly--NH2      >10                            47    MeO                                                                              val       d-leu         gly--NH2      >10                            48    MeO                                                                              phg       d-leu         gly--NH2      >10                            49    MeO                                                                              pro       d-leu         gly--NH2      >10                            50    MeO                                                                              leu       d-leu         gly--NH2      0.5                            51    MeO                                                                              leu       d-leu         gly--NH2      0.5                            52    MeO                                                                              d-leu     ala--NH2                    >10                            53    MeO                                                                              d-leu     d-ala--NH2                  >10                            54    MeO                                                                              d-leu     d-pro--NH2                  >10                            55    MeO                                                                              d-pro--NH2                            >10                            56    MeO                                                                              d-leu     d-phe--NH2                  >10                            57    MeO                                                                              d-nva     d-leu--NH2                  >10                            58    MeO                                                                              nva       d-leu--NH2                  >10                            59    MeO                                                                              d-ala     d-leu--NH2                  >10                            60    MeO                                                                              d-tic     d-leu--NH2                  >10                            61    MeO                                                                              d-ser     d-leu--NH2                  >10                            62    MeO                                                                              diisopropyl                           >10                            63    MeO                                                                              morpholine                            >10                            64    MeO                                                                              pyrrolidine                           >10                            65    MeO                                                                              phenethylamine                        >10                            66    MeO                                                                              phenpropylamine                       >10                            67    MeO                                                                              piperidine                            >10                            68    MeO                                                                              diethylamine                          >10                            69    MeO                                                                              cyclobutylamine                       >10                            70    MeO                                                                              heptylamine                           >10                            71    MeO                                                                              3-                                    >10                                     methoxypropylamine                                                   72    MeO                                                                              3,4-                                  >10                                     dimethoxypheny                                                                ethylaime                                                            73    MeO                                                                              N-                                    >10                                     methylbenzylamine                                                    74    MeO                                                                              cyclopentylamine                      >10                            75    MeO                                                                              2,6-                                  >10                                     dimethylpiperidine                                                   76    MeO                                                                              N-                                    >10                                     benzylethanolamine                                                   77    MeO                                                                              indoline                              >10                            78    MeO                                                                              dimethylamine                         >10                            79    MeO                                                                              bis(2-                                >10                                     methoxyethyl)amine                                                   80    MeO                                                                              piperonylamine                        >10                            81    MeO                                                                              4-                                    >10                                     hydroxypiperidine                                                    82    MeO                                                                              3-iodoaniline                         >10                            83    MeO                                                                              1-aminoindane                         >10                            84    MeO                                                                              ethanolamine                          >10                            85    MeO                                                                              4-                                    >10                                     methoxybenzylamine                                                   86    MeO                                                                              leu       d-nle         gly--NH2      >10                            87    MeO                                                                              leu       d-nva         gly--NH2      >10                            88    MeO                                                                              leu       d-phe         gly--NH2      >10                            89    MeO                                                                              leu       d-cha         gly--NH2      >10                            90    MeO                                                                              leu       d-val         gly--NH2      >10                            91    MeO                                                                              leu       d-phg         gly--NH2      10                             92    MeO                                                                              leu       d-pro         gly--NH2      >10                            93    MeO                                                                              d-arg     d-leu--NH2                  >10                            94    MeO                                                                              d-asp     d-leu--NH2                  >10                            95    MeO                                                                              d-asn     d-leu--NH2                  10                             96    MeO                                                                              d-asn     d-leu--NH2                  >10                            97    MeO                                                                              d-glu     d-leu--NH2                  >10                            98    MeO                                                                              d-gln     d-leu--NH2                  >10                            99    MeO                                                                              d-his     d-leu--NH2                  >10                            100   MeO                                                                              d-lys     d-leu--NH2                  >10                            101   MeO                                                                              d-thi     d-leu--NH2                  >10                            102   MeO                                                                              d-tyr     d-leu--NH2                  >10                            103   MeO                                                                              d-trp     d-leu--NH2                  >10                            104   MeO                                                                              4-                                    >10                                     (aminomethyl)                                                                 pyridine                                                             105   MeO                                                                              1,2-                                  >10                                     diaminopropane                                                       106   MeO                                                                              thiomorpholine                        >10                            107   MeO                                                                              2-                                    >10                                     methoxybenzylamine                                                   108   MeO                                                                              4-                                    >10                                     methylpiperidine                                                     109   MeO                                                                              3-pyrrolidinol                        >10                            110   MeO                                                                              4-amino-1-                            >10                                     benzylpiperidine                                                     111   MeO                                                                              3-amino-1,2-                          >10                                     propandiol                                                           112   MeO                                                                              1-(2-                                 >10                                     aminoethyl)                                                                   pyrrolidine                                                          113   MeO                                                                              2-amino-2-                            >10                                     methyl-1-                                                                     propanol                                                             114   MeO                                                                              2-                                    >10                                     (amionmethyl)                                                                 pyridine                                                             115   MeO                                                                              2-                                    >10                                     (methylamio)ethanol                                                  116   MeO                                                                              3-(3-                                 >10                                     pyridylmethylamino)                                                           propionitrile                                                        117   MeO                                                                              2-                                    >10                                     methyoxethylamine                                                    118   MeO                                                                              2-amino-1-                            >10                                     phenyl-1,3-                                                                   propanediol                                                          119   MeO                                                                              2-                                    >10                                     pyrrolidinomethanol                                                  120   MeO                                                                              3-phenyl-1-                           >10                                     propylamine                                                          121   MeO                                                                              p-anisidine                           >10                            122   MeO                                                                              aniline                               >10                            123   MeO                                                                              leu       d-leu         d-val--NH2    >10                            124   MeO                                                                              leu       d-leu         val--NH2      >10                            125   MeO                                                                              leu       d-leu         d-ala--NH2    >10                            126   MeO                                                                              leu       d-leu         ala--NH2      >10                            127   MeO                                                                              leu       d-leu         d-phe--NH2    >10                            128   MeO                                                                              leu       d-leu         phe--NH2      >10                            129   MeO                                                                              d-leu     d-arg--NH2                  >10                            130   MeO                                                                              d-leu     d-asp--NH2                  >10                            131   MeO                                                                              d-leu     d-asn--NH2                  >10                            132   MeO                                                                              d-leu     d-cha--NH2                  >10                            133   MeO                                                                              d-leu     d-glu--NH2                  >10                            134   MeO                                                                              d-leu     d-nle--NH2                  >10                            135   MeO                                                                              d-leu     d-tyr--NR2                  >10                            136   MeO                                                                              d-leu     d-trp--NH2                  >10                            137   MeO                                                                              d-leu     d-gln--NH2                  >10                            138   MeO                                                                              d-leu     d-lys--NH2                  >10                            139   MeO                                                                              d-leu     d-nva--NH2                  >10                            140   MeO                                                                              d-leu     d-phg--NH2                  >10                            141   MeO                                                                              d-leu     d-ser--NH2                  >10                            142   MeO                                                                              d-leu     d-thi--NH2                  >10                            143   MeO                                                                              d-leu     d-tic--NH2                  >10                            144   MeO                                                                              N-(4-                                 >10                                     hydroxyphenyl)-                                                               2-napthylamine                                                       145   MeO                                                                              2-amino-4,6-                          >10                                     dihydroxy-5-                                                                  methylpyrimidine                                                     146   MeO                                                                              5-                                    >10                                     (hydroxymethyl)-                                                              2-pyrrolidinone                                                      147   MeO                                                                              3-                                    >10                                     hydroxydiphenyl                                                               amine                                                                148   MeO                                                                              2-amino-4-                            >10                                     phenylphenol                                                         149   MeO                                                                              2-(4-                                 >10                                     methoxybenzylamino)                                                           pyridine                                                             150   MeO                                                                              hexamethylenediamine                  >10                            151   MeO                                                                              4-hydroxy-4-                          >10                                     phenylpiperidine                                                     152   MeO                                                                              4-iodoaniline                         >10                            153   MeO                                                                              2-methyl-6-                           >10                                     nitroaniline                                                         154   MeO                                                                              (r)-(-)-5-                            >10                                     (hydroxymethyl)-                                                              2-pyrrolidinone                                                      155   MeO                                                                              2-amino-4-                            >10                                     chloro-6-methyl-                                                              pyrimidine                                                           156   MeO                                                                              2-amino-5-                            >10                                     chloropyridine                                                       157   MeO                                                                              3,4-                                  >10                                     dichloroaniline                                                      158   MeO                                                                              4-amino-2-                            >10                                     mercaptopyrimidine                                                   159   H  leu       d-leu         gly--NH2      >10                            160   MeO                                                                              indoline                              >10                            161   MeO                                                                              phenpropyl                            >50                            162   MeO                                                                              p-anisidine                           >50                            163   MeO                                                                              piperonyl                             >50                            164   MeO                                                                              2-pyrrolidine-                        >50                                     methanol                                                             165   MeO                                                                              2-amino-1-                            >50                                     phenyl-1,3-                                                                   propanediol                                                          166   MeO                                                                              N-benzyl-                             >50                                     ethanolamine                                                         167   MeO                                                                              dimethylamide                         >50                            168   MeO                                                                              anilino                               >50                            169   MeO                                                                              bis-2-                                >50                                     methoxyethylamine                                                    170   MeO                                                                              leu       d-leu         aib--NH2      >10                            171   MeO                                                                              leu       d-leu--OH                   >10                            172   MeO                                                                              leu       d-leu--OH                   >10                            173   MeO                                                                              leu       d-leu         benzyl        0.5                            174   MeO                                                                              leu       d-leu         morpholine    >10                            175   MeO                                                                              leu       d-leu         piperdinyl    >10                            176   MeO                                                                              leu       d-leu         pyrrolidino   >10                            177   MeO                                                                              leu       d-leu         dibenzyl      1                              178   MeO                                                                              leu       d-leu         hydroxyethyl  >10                            179   MeO                                                                              leu       d-leu         N-methylbenzyl                                                                              >10                            180   MeO                                                                              leu       d-leu         N-methylbenzyl                                                                              >10                            181   MeO                                                                              aminomethylcycl                       >10                                     ohexaneamide                                                         182   MeO                                                                              leu       aminomethylcyclohexaneamide >10                            183   MeO                                                                              leu       aminomethylcyclohexaneamide                                                                 gly--NH2      >10                            184   MeO                                                                              leu       d-leu         phenyl        >10                            185   MeO                                                                              leu       d-leu         phenethyl     >10                            186   MeO                                                                              leu       d-leu         phenpropyl    >10                            187   MeO                                                                              leu       d-leu         indane        0.5                            188   MeO                                                                              leu       d-leu         aminomethylcyclohexane                                                                      >10                            189   MeO                                                                              leu       d-leu         aminomethylpyridine                                                                         >10                            190   MeO                                                                              leu       d-leu         adamantyl     >10                            191   MeO                                                                              leu       d-leu         tetrahydroisoquinoline                                                                      >10                            192   MeO                                                                              leu       d-leu         4-pyridylmethyl                                                                             10                             193   MeO                                                                              leu       d-leu         N-benzylhydroxamic                                                                          0.4                            194   MeO                                                                              leu       d-leu         4-methoxybenzyl                                                                             >10                            195   MeO                                                                              leu       d-leu         4-nitrobenzyl 10                             196   MeO                                                                              leu       d-leu         2,6-difluorobenzyl                                                                          0.08                           197   MeO                                                                              leu       d-leu         piperonyl     0.2                            198   MeO                                                                              d-leu     benzyl                      >10                            199   MeO                                                                              d-leu     dibenzyl                    >10                            200   MeO                                                                              d-leu     isoamyl                     >10                            __________________________________________________________________________

EXAMPLE 3

Compounds prepared according to the method of Example 1 were assayedagainst several different cell lines. Cell monolayers were cultured inthe presence of test compound for 18 hours to access their ability toinhibit cell proliferation. Cell proliferation was determinedcolorimetrically using the Celltiter 96 Aqueous non-radioactive cellproliferation assay (Promega) where cell proliferation is directlyproportional to absorbance at 490 nm. Results are quoted as the IC₅₀ inμg/ml for inhibition of cell proliferation in various cell types.

    ______________________________________                                        Compound                                                                              RAW      MCF-7   OVCAR   CaCo Panc-I                                  ______________________________________                                        173     20       10      8       17   17                                      187     10       6       5       8    7                                       194     1                3            8                                       196     7                2            8                                       197     12               2            8                                       ______________________________________                                    

EXAMPLE 4

Compounds prepared according to the method of Example 1 were tested forthe inhibition of LPS induced TNF synthesis. RAW cells were pretreatedwith different concentrations of test compound for 1 hour prior to theadministration of lipopolysaccharide (100 ng/mi). Cell culturesupernatants were harvested after 1 hour and assayed for TNFconcentration by ELISA (Biosource)

    ______________________________________                                               Compound                                                                              IC.sub.50  (μg/ml)                                          ______________________________________                                               173     5                                                                     187     5                                                                     194     3                                                                     196     3                                                                     197     3                                                              ______________________________________                                    

EXAMPLE 5

This Example examines the ability of compound 173 and particularlycompound 187 described in Tables 1 & 2 above to inhibit proteasomeactivity as indicated, in part, by the presence of IκB and/or p105 ininhibited cells. In order for NF-κB to translocate to the nucleus inresponse to a stimulus such as lipopolysaccharide (LPS) and activatetranscription two proteolytic events need to occur, namely degradationof the inhibitory protein IκB and processing of p105 to p50. Theseproteolytic events serve to unmask the nuclear localization signal ofNF-κB.

Inhibition of LPS-induced IκB Degradation

RAW cells were pretreated with different concentrations of test compoundfor 1 hour prior to the administration of lipopolysaccharide (100ng/ml). Whole cell lysates were harvested after 1 hour, 10 μg, proteinwas separated by SDS-PAGE, transferred to nitrocellulose and assayed forimmunoreactivity with anti-IκBa antibody. Western blots, (See FIG. 1),were visualized using the Boehringer Mannheim Chemiluminescent detectionkit. The blot shows that IκB is present in cells treated with as littleas 5 μg/ml of compounds 173 and 187.

Inhibition of LPS-induced p150 to p50 Processing

Compound 187 as described in Tables 1 & 2 above was used to pretreat RAWcells as described above, and whole cell lysates prepared as describedabove were analyzed for immunoreactivity toward an anti-p50 antibody.The results, set forth in FIG. 2 indicate that p50 and p150 are bothpresent in cells treated with as little as 5 μg/ml of compound 187,while in untreated cells, majority of P 105 has been processed to p50.

Inhibition of LPS-induced Translocation of NF-κB to the Nuclear Fractionof the Cell

RAW cells were treated for 1 hour with compound 187 (20 μg/ml) and thenincubated with lipopolysaccharide (100 ng/ml) for a further one hour.Nuclear fractions were prepared according to standard procedures.Binding reactions for gel mobility shift assays contained 5 μg ofnuclear extract protein, 50,000 cpm of ³² P-labeled NF-κB consensusbinding oligonucleotide in the presence and absence of a fifty foldexcess of unlabeled oligonucleotide. The gel mobility shift assay, setforth in FIG. 3 shows that compound 187 is effective in inhibiting theaccumulation of NF-κB in the nucleus of cells.

What we claim is:
 1. A method for treating mammals with cellproliferation disorders, susceptible to treatment with the compoundsherein, comprising administering to the mammal a therapeuticallyeffective amount of a compound having the formula: ##STR9## wherein R₁,R₂, R₄, R₅, R₆, R₇, R₈ and R₉ are each hydrogen, R₃ is MeO, X is:##STR10## wherein D₁ is leu, D₂ is d-leu, or d-leu-OH, and E is selectedfrom hydrogen, benzyl, indane, 4-methoxybenzyl, 2,6-difluorobenzyl, andpiperonyl.
 2. The method of claim 1 wherein D₂ is d-leu.
 3. The methodof claim 2 wherein E is selected from benzyl, indane, 4-methoxybenzyl,2,6-difluorobenzyl, and piperonyl.
 4. The method of claim 3 wherein E is2,6-difluorobenzyl.
 5. The method according to claim 1 wherein themammal is a human.
 6. The method according to claim 1 wherein thetherapeutically effective amount ranges from about 0.001 to about 100mg/kg weight of the mammal.
 7. The method of claim 1 wherein thecomposition is administered to a mammal suffering from a cellproliferation disorder selected from the group consisting of cancer,restenosis, host graft disease, and gout.
 8. The method of claim 7wherein the cell proliferation disorder is restenosis.
 9. The method ofclaim 7 wherein the cell proliferation is disorder cancer.
 10. Themethod according to claim 7 wherein the therapeutic agent inducesapoptosis.
 11. The method of claim 7 wherein the cell proliferationdisorder is polycystic kidney disease.